Nutritional copper regulates the activity levels of lysyl oxidase, a copper-metalloenzyme in aorta and other connective tissues. This discovery has opened the way to meaningful studies of how specific trace metals control biochemical events in cells; specifically, the biosynthesis of a metalloenzyme. The present proposal intends to move laterally from the basic observation and investigate the possible role for serum and intracellular copper-binding proteins in the mechanism. Further probes will attempt to identify the subcellular site of copper incorporation into the aorta enzyme. The studies will use normal animals whose dietary intake of copper has been strictly controlled. Changes in aortic lysyl oxidase activity will be correlated with the changes in serum copper proteins, ultimately to the one specific protein that is needed in the activation mechanism. Closer biochemical studies will use aortic tissue suspended in a chemically defined medium with radioactive copper present to assist in the identification of important intermediates. Finally, with the crucial factors known, an attempt will be made to reconstruct the events associated with the transfer of copper into the enzyme using a cell-free system and components identified earlier. Such a system will be helpful in further investigations of the energy requirements and additional factors which may be involved.